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Biocatalytic versatility of engineered and wild-type tyrosinase from R. solanacearum for the synthesis of 4-halocatechols.
Davis, Reeta; Molloy, Susan; Quigley, Blathnaid; Nikodinovic-Runic, Jasmina; Solano, Francisco; O'Connor, Kevin E.
Afiliación
  • Davis R; School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland.
  • Molloy S; School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland.
  • Quigley B; School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland.
  • Nikodinovic-Runic J; School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland.
  • Solano F; Institute for Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, Belgrade, 11000, Serbia.
  • O'Connor KE; Department of Biochemistry and Molecular Biology B and Immunology, University of Murcia, Murcia, Spain.
Appl Microbiol Biotechnol ; 102(12): 5121-5131, 2018 Jun.
Article en En | MEDLINE | ID: mdl-29691629
We evaluated the kinetic characteristics of wild type (WT) and three engineered variants (RVC10, RV145, and C10_N322S) of tyrosinase from Ralstonia solanacearum and their potential as biocatalysts to produce halogenated catechols. RV145 exhibited a 3.6- to 14.5-fold improvement in catalytic efficiency (kcat/Km) with both reductions in Km and increases in kcat compared to WT, making it the best R. solanacearum tyrosinase variant towards halogenated phenols. RVC10 also exhibited increases in catalytic efficiency with all the tested phenols. A single-mutation variant (C10_N322S) exhibited the greatest improvement in kcat but lowest improvement in catalytic efficiency due to an increase in Km compared to WT. Consistent with kinetic characteristics, biotransformation experiments showed that RV145 was a superior biocatalyst in comparison to WT. To prevent through conversion of the catechol to quinone, ascorbic acid (AA) was added to the biotransformation medium in 1:2 (substrate:AA) ratio resulting in a catechol yield of > 90%. Flask experiments with 10 mM 4-iodophenol and 10 µg/mL of the RV145 enzyme yielded 9.5 mM 4-iodocatechol in the presence of 20 mM AA in 30 min. Similarly, 10 mM 4-fluorophenol was completely consumed by 20 µg/mL of RV145 enzyme and yielded 9.2 mM 4-fluorocatechol in the presence of 20 mM AA in 80 min. The biotransformation of 20 mM 4-fluorphenol was incomplete (93%) and the yield of 4-flurocatechol was 87.5%. The 4-halophenol conversion rates and product yields obtained in this study are the highest reported using tyrosinase or any other enzyme.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Catecoles / Monofenol Monooxigenasa / Ralstonia solanacearum Idioma: En Revista: Appl Microbiol Biotechnol Año: 2018 Tipo del documento: Article País de afiliación: Irlanda Pais de publicación: Alemania
Texto completo
Añadir a Mi BVS
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XML
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Catecoles / Monofenol Monooxigenasa / Ralstonia solanacearum Idioma: En Revista: Appl Microbiol Biotechnol Año: 2018 Tipo del documento: Article País de afiliación: Irlanda Pais de publicación: Alemania